Treatment of human viral infections

ABSTRACT

Treatment of AIDS or humans carrying or infected with the AIDS virus or having antibodies to the AIDS virus is disclosed using the compound 3&#39;-azido-3&#39;-deoxythymidine or a pharmaceutically acceptable basic salt thereof. 
     Also disclosed is the use of the 5&#39;-mono-, di- and triphosphate of 3&#39;-azido-3&#39;-deoxythymidine or a pharmaceutically acceptable basic salt thereof for the same purpose.

This is a continuation of copending application Ser. No. 07/407,579filed on Sept. 15, 1989, now abandoned, which is a continuation ofcopending application Ser. No. 07/153,258, filed on Feb. 8, 1988, nowU.S. Pat. No. 4,874,609, which is a continuation of Ser. No. 06/776,899,filed Sept. 17, 1985, now U.S. Pat. No. 4,724,232.

The acquired immunodeficiency syndrome (AIDS) is considered to be causedby the HTLV III virus (also identified as HTLV III/LAV). HTLV III is anRNA genetically unique retrovirus, having a gene not found to date inother retroviruses, which allows it to rapidly replicate in a human hostcell. The HTLV III virus appears to preferentially attack helper T-cells(T-lymphocytes or OKT4-bearing T-cells as they are sometimes known) andpossibly other human cells, e.g., certain cells within the brain. Thehelper T-cells are invaded by the virus and the T-cell becomes an HTLVIII virus producer. The helper T-cells are quickly destroyed and theirnumber in the human being is depleted to such an extent that the body'sB-cells as well as other T-cells normally stimulated by helper T-cellsno longer function normally or produce sufficient lymphokines andantibodies to destroy the invading virus or other invading microbes,etc.

While the HTLV III virus does not necessarily cause death per se, itdoes in many cases cause the human's immune system to be so severelydepressed that the human falls prey to various other diseases (secondaryinfections or unusual tumors) such as herpes, cytomegalovirus, Kaposi'ssarcoma and Epstein-Barr virus related lymphomas among others. Thesesecondary infections are separately treated using other medications asis conventional. In some humans infected with HTLV III virus,(hereinafter referred to the AIDS virus and which is meant herein toinclude mutants thereof) humans seem to live on with little or nosymptoms, but appear to have persistent infections. Another group ofhumans suffers mild immune system depression with various symptoms suchas weight loss, malaise, fever, swollen lymph nodes. These syndromeshave been called persistent generalized lymphadenopathy syndrome (PGL)and AIDS related complex (ARC) and may or may not develop into AIDS.

In all cases, those infected with the AIDS virus (HTLV III virus) arebelieved to be persistently infective to others.

It has now been discovered that the compound known as3'-azido-3'-deoxythymidine having the formula I ##STR1## is useful intreating humans identified as having AIDS as well as humans identifiedas infected by or carrying the AIDS virus or having antibodies to theAIDS virus in order to inhibit the acquired immunodeficiency syndromefrom developing in such humans or being transmitted to others. Testswith the compound of formula I in humans has shown that the amount ofAIDS virus is significantly reduced at least transiently, the number ofhelper T-cells, T-cells and platelets are increased over those beforetreatment and in two cases to date the immune system appears to havebeen reestablished (restored) in so far as the human patient's abilityto mount a delayed hypersensitivity skin test immune response wasrestored.

The compound of formula I may be administered per se or in the form of apharmaceutically acceptable salt, e.g., an alkali metal salt such assodium or potassium, an alkaline earth salt or an ammonium salt (all ofwhich are hereinafter referred to as a pharmaceutically acceptable basesalt).

The salts of the compound convert to the compound per se after beingadministered to the human and are thus prodrugs. The compound3'-azido-3'-deoxythymidine penetrates into the infected cells aftercontacting same and is converted by the enzymes of cells such as Tlymphocytes, astroglia cells, macrophages and others to themonophosphate thereof. The monophosphate is then converted by thecellular enzymes to the diphosphate of the compound of formula I andultimately to the triphosphate of the compound of formula I.

In human cells infected with the human aids virus the mono- andtriphosphate of the compound of formula I act as inhibitors of the viralreverse transcriptase necessary for viral replication. All prodrugs(precursors) are administered to a human in an amount sufficient togenerate an effective amount of the compound which contacts the AIDSvirus and interacts with it (e.g., prevents replication thereof).

Thus the compound of formula I can be said to be a pro-drug afterentering the human T-lymphocytes or other human cells since it is anintermediate (precursor) to the mono-, di- and triphosphate thereof.

It is believed that the mono-, di- and triphosphates of the compound offormula (I) can also be said to be prodrugs since they would invariably(at least in part) be hydrolized in the body to3'-azido-3'-deoxythymidine which is taken up by the cells.

As another feature of this invention, there is also disclosed the methodof administering to a human in need thereof the mono-, di-, ortriphosphate of the compound of formula I or their pharmaceuticallyacceptable base salts (i.e., alkali metal, alkaline earth or ammoniumsalt) to treat AIDS, to inhibit the replication of the AIDS virus ininfected human cells and to prevent AIDS from developing in humansinfected with the AIDS virus or carrying antibodies to the AIDS virus.The mono-, di- and triphosphates of 3'-azido-3'-deoxythymidine are ofthe formulas II, III and IV respectively. ##STR2##

The present invention also discloses compounds of formulas I, II, IIIand IV and their appropriate salts for use in the treatment of theconditions referred to above, as well as the use of such compounds inthe preparation of pharmaceutical formulations for the treatment of suchconditions. The above mentioned pharmaceutically acceptable salts may beprepared in a conventional manner, e.g., treatment of the compound withan appropriate base.

In general for the above AIDS virus infections, a suitable effectivedose of the 3'-azido-3'-deoxythymidine compound, its pharmaceuticallyacceptable basic salts, its mono-, di- or triphosphates or theirpharmaceutically acceptable basic salts (all of which are herein afterreferred to as the administered ingredient) will be in the range 5 to250 mg per kilogram body weight of recipient per day, preferably in therange of 7.5 to 100 mg per kilogram body weight per day and mostpreferably in the range 10 to 40 mg per kilogram body weight per day. Ina clinical program in treating humans with AIDS, the compound3'-azido-3'-deoxythymidine is being administered as an infusion (IV) at1 and 2.5 mg/kilogram every eight hours. The desired dose is preferablypresented as two, three, four or more sub-doses administered atappropriate intervals throughout the day. These sub-doses may beadministered as unit dosage forms, for example, containing 5 to 500 mg,preferably 10 to 200 mg and most preferably 20 to 100 mg of activeingredient per unit dosage form.

Administration may be by any suitable route including oral, rectal,nasal, topical (including buccal and sublingual), vaginal and parenteral(including subcutaneous, intramuscular, intravenous and intradermal)with oral or parenteral being preferred. It will be appreciated that thepreferred route may vary with, for example, the condition and age of therecipient.

A preferred dose is administered to achieve peak plasma concentrationsof the compound of formula I, its mono-, di- or triphosphate or itspharmaceutically acceptable salts of from about 1 to about 100 μM,preferably about 5 to 80 μM, most preferably about 7.5 to about 50 μM.This may be achieved, for example, by the intravenous injection of asterile 0.1 to 5% solution of the administered ingredients in saline asa bolus containing about 1 to about 80 mg/kg of the active ingredient.Desirable blood levels may be maintained by a continuous infusion toprovide about 0.01 to about 0.8 mg/kg/hour or by intermittent infusionsproviding about 0.04 to about 20 mg/kg of the administered ingredient.

The administered ingredients may be used in therapy in conjunction withother medicaments such as9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine,9-(2-hydroxyethoxymethyl)guanine (acyclovir),2-amino-9-(2-hydroxyethoxymethyl)purine, suramin, ribavirin,antimoniotungstate (HPA-23), interferon, e.g., α interferon, interleukinII, and phosphonoformate (Foscarnet) or in conjunction with other immunemodulation including bone marrow or lymphocyte transplants or othermedications such as levamisol or thymosin which would increaselymphocyte numbers and/or function as is appropriate.

The compound of formula I, 3'-azido-3'-deoxythymidine, (sometimes alsoreferred as azidothymidine) is disclosed in J. R. Horwitz et al, J. Org.Chem. 29, July 1964, pp. 2076-2078; M. Imazawa et al, J. Org. Chem.,43(15) 1978, pp. 3044-3048; also see Biochemical Pharmacology, Vol. 29,pp. 1849-1851; C. J. Kreig et al, Experimental Cell Research 116 (1978)pp. 21-29. Also see W. Ostertag et al, Proc. Nat. Acad. Sci. USA 71(1974) for some of its biological activities.

While it is possible for the administered ingredients to be administeredalone, it is preferable to present them as part of a pharmaceuticalformulation. The formulations of the present invention comprise at leastone administered ingredient, as above defined, together with one or moreacceptable carriers thereof and optionally other therapeuticingredients. The carrier(s) must be "acceptable" in the sense of beingcompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal)administration. The formulations may conveniently be presented in unitdosage form, e.g., tablets and sustained release capsules, and may beprepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the to beadministered ingredients with the carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and thenif necessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Formulations suitable for topical administration in the include lozengescomprising the ingredients in a flavoured basis, usually sucrose andacacia or tragacanth; pastilles comprising the active ingredient in aninert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered and a pharmaceutically acceptable carrier.A preferred topical delivery system is a transdermal patch containingthe ingredient to be administered.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration wherein the carrier is asolid include a coarse powder having a particle size, for example, inthe range 20 to 500 microns which is administered in the manner in whichsnuff is taken, i.e., by rapid inhalation through the nasal passage froma container of the powder held close up to the nose. Suitableformulations wherein the carrier is a liquid, for administration, as forexample, a nasal spray or as nasal drops, include aqueous or oilysolutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude flavouring agents.

The LD50 for 3'-azido-3'-deoxythimidine in mice and rats was found to begreater than 750 mg/kg.

The following examples illustrate the invention.

EXAMPLE 1 Preparation of 2,3'-Anhydrothymidine

Thymidine (85.4 g; 0.353 mol) was dissolved in 500 ml dry DMF(dimethylformamide) and added toN-(2-chloro-1,1,2-trifluoroethyl)dielthylamine (100.3 g; 0.529 mol)[prepared according to the method of D. E. Ayer, J. Med. Chem. 6, 608(1963)]. This solution was heated at 70° C. for 30 minutes then pouredinto 950 mL ethanol with vigorous stirring. The product precipitatedfrom this solution and was filtered. The ethanol supernatant wasrefrigerated then filtered to yield a total of 47.75 g (0.213 mol;60.3%) of 2,3'-anhydrothymidine mp=228°-230° C.

EXAMPLE 2 Preparation for 3'-Azido-3'-deoxythymidine

2,3'-Anhydrothymidine (25 g; 0.1115 mol) and NaN3 (29 g; 0.446 mol) wassuspended in a mixture of 250 mL DMF and 38 mL H₂ O. The reaction wasrefluxed for 5 hours at which time it was poured into 1 liter of H₂ O.This aqueous solution was extracted with ethyl acetate (EtOAc) (3×700ml). The EtOAc was dried over Na₂ SO₄, filtered, and then EtOAc wasremoved in vacuo to yield a viscous oil. This oil was stirred with 200mL water resulting in a solid, 3'-azido-3'-deoxythymidine, 9.15 g(0.0342 mol; 30.7%) mp=116°-118° C.

EXAMPLE 3 Preparation of Sodium Salt of 3'-Azido-3'-deoxythymidine

Approximately one gram of 3'-azido-3'-deoxythymidine was dissolved in 50mL of distilled water. The pH was adjusted to 12 with 1N NaOH.Approximately half of the solution was freeze dried. A white powder, thesodium salt of 3'-azido-3'-deoxythymidine on the 0.6 hydrate, 0.415 g,resulted.

Analysis calculated for C₁₀ H₁₂ N₅ NaO₄.6/10 H₂ O. Calculated: C,40.03;H,4.43; N,23.34; Na,7.66. Found: C,39.88; H,4.34; N,23.29; Na,7.90.

EXAMPLE 4 Preparation of 5'-Monophosphate of 3'-Azido-3'-deoxythymidine

3'-Azido-3'-deoxythymidine (0.5 g, 1.87 mmol) was dissolved in 5 mL oftriethyl phosphate and the mixture was cooled to -5° C. Phosphorusoxychloride (0.685 mL, 7 mmol) was added in one portion to the rapidlystirred solution which was then maintained at -10° C. for 22 hours. Analiquot was removed and added to concentrated ammonium hydroxide.Analysis of this sample on TLC (cellulose, n-PrOH:H2O, 7:3 v/v) showedno remaining starting material and a single fluorescent spot with lowermobility than the nucleoside. The reaction mixture was poured onto 20 mLof ice and water. This was placed in an ice bath and the pH of thesolution was adjusted to a value of 7.5 by the addition of 2N NaOH. Thebasic mixture was extracted once with chloroform and once with ether.The aqueous layer was again adjusted to give a pH of 7.5 andconcentrated in vacuo to remove residual organic solvent. The materialwas stored at -10° C. until purified as follows:

Deactivated charcoal was prepared by washing coconut charcoal (50-200mesh, 100 g) with 500 mL of 1N HCl, 3 L of water, 35 mL of 3% toluene in95% ethanol, 600 mL of 95% ethanol and finally extensively with water.Deactivated charcoal (12 mL of settled wet charcoal) was added withstirring to the monophosphate solution (0.72 g, 1.8 mmol, 30 mL). Thesupernatant was decanted and the charcoal was washed with 150 mL ofwater. The nucleotide was eluted from the charcoal by washing with 120mL of 1.5M ammonium hydroxide in 50% ethanol. This solution was filteredthrough a 0.22 micron filter, concentrated in vacuo to 10 mL, filteredthrough a Amicon Centriflo CF-25 membrane, and lyophilized. The yield ofdiammonium 3'-azido-3'-deoxythymidine-5'-monophosphate was 0.36 g (0.94mmol, 52%). This compound was characterized as a nucleoside5'-monophosphate by the ability of 5'-nucleotidase to degrade it to thenucleoside.

EXAMPLE 5 Preparation of the 5'-Di- and Triphosphate of3'-Azido-3'-deoxythymidine

The di- and triphosphate of 3'-azido-3'-deoxythymidine were preparedfrom the ammonium salt of the 5'-monophosphate of3'-azido-3'-deoxythymidine by a four step sequence.

Step I--Preparation of Bis-(Tri-n-butylammonium) Pyrophosphate

A column of DOW 50 pyridinium resin was prepared by pouring 40 mL ofresin into a 25 cm diameter column and washing with water until no morecolor eluted. Sodium pyrophosphate decahydrate (1.12 g, 2.51 mmol) wasdissolved in 30 mL of water and applied to the column. The column waseluted with water. A 125 mL fraction of the eluant which contained UVabsorbing material was collected. The volume was reduced to 10 mL invacuo and tri-n-butylamine (1.2 mL) was added. The volume was reduced invacuo and the residue was dried by coevaporation with pyridine fourtimes. The product was stored in a freezer (-5° C.).

Step II--Preparation of the Acid Form of the Monophosphate of3'-Azido-3-deoxythymidine

The acid form of the monophosphate was prepared by passing the ammoniumsalt (0.1 g, 0.283 mmol) dissolved in 6 mL of water, through a 1.5 mL(10 eq.) column of DOW 50 H⁺.

Step III--Preparation of Phosphoromorpholidate Derivative

The hydrogen form of the monophosphate, 0.283 mmol, was dissolved in 9mL of water. Morpholine (99 μL, 1.13 mmol, 4 eq.) was added and thesolution heated to reflux. Dicyclohexyl carbodiimide (0.234 g, 1.13mmol, 4 eq.) dissolved in t-butanol (5 mL) was added over a three-hourperiod. The reaction was refluxed overnight. The reaction was cooled toroom temperature, filtered, and the solvents removed in vacuo. Ethanolwas added and evaporated in vacuo four times. The residue was dissolvedin methanol and the phosphoromorpholidate precipitated by the additionof ether. The precipitate was triturated with ether four times and driedon a rotary evaporator. A weight yield of 97 mG, 50%, was obtained.

Step IV--Preparation of the 5'-Di- and Triphosphates of3'-Azido-3'-deoxythymidine

The phosphoromorpholidate derivative was dried by removal of pyridine invacuo four times. The bis-(tri-n-butylammonium) pyrophosphate was alsodried by removal of pyridine in vacuo. The phosphoromorpholidate wasdissolved in pyridine, 5 mL, and added to the vessel containing thepyrophosphate reagent. The reaction was allowed to continue overnight atroom temperature. The pyridine was removed in vacuo. Water was added tothe residue and removed in vacuo three times. The residue was frozen.

The reaction mixture (0.09 g) was thawed and dissolved in 50 mL ofwater. The solution was applied to a column (1×10 cm) of DEAE SaphadexA-25 which had been equilibrated 50 mM ammonium bicarbonate. Thephosphates were eluted with a 300 mL linear gradient of 50-800 mMammonium bicarbonate. The fractions containing the diphosphatenucleotide were pooled as were those containing the triphosphatenucleotide. The pooled diphosphate and triphosphate fractions were eachdried in vacuo, redissolved in water, dried again, redissolved in waterand lyophilized. The yields were: the diphosphate as the triammoniumsalt, 0.014 g; the triphosphate, as the tetrammonium salt, 0.002 g.

EXAMPLE 6 Enzymatic Synthesis of the 5'-Triphosphate of3'-Azido-3'-deoxythymidine

The 5'-triphosphate was synthesized from the 5'-diphosphate usingpyruvate kinase and nucleoside diphosphate kinase. The reaction mixturecontained: 6 mM 3'-azido TDP, 12 mM adenosine triphosphate, 40 mM MgCl2,40 mM potassium piperazine-N,N'-bis(2-ethanesulfonic acid) PIPES buffer,pH 6.8), 30 mM phosphoenolpyruvate, 40 IU/ml nucleoside diphosphatekinase and 100 IU/ml pyruvate kinase in a final volume of 5 mL. Thereaction mixture was incubated at 37° C. for 5 days. The reactionmixture was applied to a column (2.5×10 cm) of DEAE Saphadex A-25 whichhad been equilibrated with ammonium bicarbonate. The nucleotides wereeluted with a gradient of 100-1000 mM ammonium bicarbonate. Fractionscontaining the triphosphate were pooled, and evaporated to dryness invacuo. The compound was further purified using a preparative HPLC column(Whatman, Inc., Magnum 9 SAX) eluted with a gradient of 10-1000 mMpotassium phosphate, ph 3.5. The resulting compound was further purifiedusing a DEAE Saphadex A-25 column as above. The fractions containing thetetrammonium 3'-azido-3'-deoxythymidine-5'-triphosphate were pooled,dried in vacuo, redissolved in water and lyophilized to yield 0.008 g(0.01 mmol).

EXAMPLE 7

    ______________________________________                                        Tablet Formulation                                                            ______________________________________                                        3'-Azido-3'-Deoxythymidine                                                                        100 mg                                                    Lactose             200 mg                                                    Starch               50 mg                                                    Polyvinylpyrrolidone                                                                               5 mg                                                     Magnesium stearate   4 mg                                                                         359 mg                                                    ______________________________________                                    

Tablets were prepared from the foregoing ingredients by wet granulationfollowed by compression.

EXAMPLE 8 Capsule

A two part soft gelatin is prepared by placing the tablet formulation ofExample 7 in a two part capsule.

EXAMPLE 9

    ______________________________________                                        Sterile Injectable Formulation                                                ______________________________________                                        3'-Azido-3'-deoxythymidine 0.125  g                                           Sterile, pyrogen-free, pH 7 phosphate buffer, q.s. to                                                    25     ml                                          ______________________________________                                    

The formulation is sterilized by heat and then placed in a sealedcontainer, e.g., glass so that it may be administered by infusion or byinjection.

EXAMPLE 10

The acid forms of the 5'-mono, di- and triphosphate nucleotides of3'-azido-3'-deoxythymidine are prepared by passing the correspondingammonium salts through DOW 50 H+ columns and lyphilizing the solutionsobtained thereby.

We claim:
 1. A method of treating a human having an HTLV III infection,which comprises administering to said human an effective amount of3'-azido-3'-deoxythymidine or a pharmaceutically acceptable salt thereofand interferon.
 2. A method according to claim 1, in which3'-azido-3'-deoxythymidine is administered orally or parenterally.
 3. Amethod according to claim 2, in which the interferon is α-interferon. 4.In a method of treating a human having an HTLV III infection and who isbeing administered α-interferon, the step of also parenterally or orallyadministering to said human an effective treatment amount of3'-azido-3'-deoxythymidine to said human.